Mutant DNA encoding insulin receptor substrate 1

ABSTRACT

A DNA sequence encoding insulin receptor substrate 1 (IRS-1), the DNA sequence containing a mutation of at least one nucleotide, and the protein encoded by said DNA sequence.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT/DK94/00227 filed Jun. 10,1994, which is incorporated herein by reference.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT/DK94/00227 filed Jun. 10,1994, which is incorporated herein by reference.

FIELD OF INVENTION

The present invention relates to a mutant DNA sequence encoding insulinreceptor substrate 1, a method of detecting a mutation in the geneencoding insulin receptor substrate 1, as well as a diagnosticcomposition and a test kit for use in the method.

BACKGROUND OF THE INVENTION

Non-insulin-dependent diabetes mellitus (NIDDM) is a common endocrinedisorder and a considerable body of evidence strongly suggests thatgenetic factors contribute to the pathogenesis (1). Studies of patientswith overt NIDDM and of individuals at high risk of NIDDM revealabnormalities of both insulin secretion and insulin action (2). However,a genetic defect at one or more loci in the cellular action of insulinand insulin-like growth factor 1 (IGF1) might well involve both thebiochemical pathways of tissues that regulate insulin secretion andinsulin sensitive hepatic and extrahepatic tissues that produce orextract glucose.

Although more than twenty different mutations of the insulin receptorgene have been reported in syndromes of severe insulin resistancefrequently associated with the skin disorder acanthosis nigricans orovarian hyperandrogenism (17), mutations in the insulin receptormolecule do not explain the genetic etiology of the common form ofNIDDM.

The common form of late onset NIDDM is a heterogeneous disorder where atleast two major defects contribute to the pathophysiology of thephenotype: insulin resistance and insulin deficiency (2). Most likelyNIDDM is also polygenic and it is suggested that subsets of patientswill display changes in various genes, in aggregate accounting for theinherited components of the disorder. The high cumulative risk ofdiabetes in offspring of NIDDM parents (30-50%) and the high concordancerate in identical twins (70-100%) underscore the significance of thegenetic etiology of the disease (1).

Insulin initiates its cellular effects by binding to the a subunit ofits tetrameric plasma membrane receptor (3). The kinase in the β subunitis thereby activated which in turn catalyzes the intramolecularautophosphorylation of specific tyrosine residues of the β subunit,further stimulating the tyrosine kinase activity of the receptor towardsother protein substrates in the cascade of insulin action.

Recently, the first endogeneous substrate for the insulin receptorkinase (termed insulin receptor substrate 1, abbreviated to IRS-1) wascloned and sequenced (4-6). The complementary DNA sequence encodes acytoplasmic, hydrophilic protein of a relative molecular mass between165 and 185 kD (27,28) which contains multiple phosphorylation sites.Besides being a substrate for the insulin receptor kinase, IRS-1 is alsophosphorylated following the activation of the IGF1 receptor kinase(16).

IRS-1 is barely detectable in cells expressing few insulin receptors,but is strongly detected in cells expressing high levels of receptorsand weakly detected in cells expressing mutant receptors defective inbiological signalling (27,28). IRS-1 is a unique molecule containing 20tyrosine phosphorylation consensus sequences, 6 of which appear in YMXM(Tyr-Met-X-Met) motifs. Following insulin stimulated tyrosinephosphorylation of YMXM motifs in the IRS-1 molecule, the phosphorylatedIRS-1 binds phosphatidylinositol 3-kinase (PI3-kinase) suggesting thatIRS-1 acts as a multisite "docking" protein to bind signal proteinsthereby linking the receptor kinase to insulin sensitive transportersand enzymes (7-15). The PI3-kinase is composed of at least two subunitsincluding a 110 kDa catalytic subunit and a 85 kDa regulatory proteinwhich contains src homology 2 domains that mediate protein-proteininteractions by binding to phosphotyrosine residues in various proteins(7-15). Interestingly, it has been demonstrated that insulin causes theinteraction between IRS-1 and PI-3 kinase via phosphorylated YMXM motifsof IRS-1 and src homology 2 domains of the 85 kDa regulatory subunit ofPI-3 kinase. Moreover, overexpression of IRS-1 potentiates theactivation of PI3-kinase in insulin stimulated cells, and tyrosylphosphorylated IRS-1 or synthetic peptides containing phosphorylatedYMXM motifs activate PI3-kinase in vitro. Besides being a substrate ofthe insulin receptor kinase, IRS-1 is also phosphorylated following theactivation of the IGF1 receptor kinase (16). Hence, it has beensuggested that IRS-1 by binding and regulating enzymes containing srchomology 2 domains may play a critical role to select and differentiatethe effects of insulin and IGF1 from those of other tyrosine kinases andto generate diversity and amplification of signal transmission intomultiple intracellular pathways (7-16).

SUMMARY OF THE INVENTION

According to the present invention, it has surprisingly been found thata number of NIDDM patients carry mutations in the gene coding for IRS-1.It is at present assumed that one or more of the mutations may beinvolved in or associated with the etiology of NIDDM, and their presencemay therefore be diagnostic for NIDDM and possibly also other disordersresulting from insulin resistance.

Accordingly, the present invention relates to a DNA construct comprisinga DNA sequence encoding insulin receptor substrate 1 (IRS-1), the DNAsequence containing a mutation of at least one nucleotide, or comprisinga fragment of the DNA sequence including said mutation.

It is at present assumed that mutation of the IRS-1 gene may beindicative of abnormalities significant for the development of NIDDM orother disorders. For instance, the mutation may give rise to thesubstitution of an amino acid in IRS-1 which may cause changes in thetertiary structure of IRS-1. Such changes may interfere with the normalinteraction between the insulin or IGF-1 receptor kinase and one or moreintracellular proteins regulating cellular metabolism and growth. Theseproteins typically have src homology 2 domains and include phosphatidylinositol 3 kinase, GRB-2 and SHPTP-2 (vide M. F. White et al., Exp.Clin. Endocrinol. 101, Suppl. 2, 1993, pp. 98-99, in particular FIG. 1).Mutations may also interfere with the transcription or translation ofthe gene, or with the stability of the IRS-1 transcript. Alternatively,the mutation may be associated with (i.e. genetically linked with) themutation which causes the disease.

In another aspect, the present invention relates to a living systemcontaining a DNA construct of the invention and capable of expressingIRS-1 wherein at least one amino acid is substituted. The living system,which may comprise a cell or a multicellular organism containing theappropriate signal transduction pathway, may be used to screen forsubstances which have an effect on insulin or IGF-1 stimulated signaltransduction in the system.

In a further aspect, the present invention relates to a method ofdetecting the presence of a mutation in the gene encoding IRS-1, themethod comprising obtaining a biological sample from a subject andanalysing the sample for a mutation of at least one nucleotide. Based oncurrent knowledge, it is assumed that the present method may be used todiagnose predisposition to NIDDM in a subject as well as other disordersresulting from insulin resistance (such as android obesity, essentialhypertension, dyslipidemia or atherosclerosis). Biological samples may,for instance, be obtained from blood, serum, plasma or tissue. Theinvention further relates to a diagnostic composition and a test kit foruse in the method.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows mutation screening using single stranded conformationpolymorphism technique (SSCP) of nucleotides 3338-3600 of the humaninsulin receptor substrate 1 (IRS-1 gene). The fragment wasPCR-amplified from genomic DNA using specific oligonucleotide primersand ³² P-labeled and subjected to nondenaturing gel electrophoresis asdescribed in Methods. The autoradiogram shows the migration profiles ofsingle stranded (ss DNA) as well as double stranded DNA (ds DNA). Lane 3depicts the migration profile of an individual who is heterozygous (He)for the glycine⁹⁷² mutation. Lanes 1 and 2 show the migration profilefor the corresponding wild type (Wt).

FIG. 2 shows direct nucleotide sequencing of a part of the 3338-3600base pair fragment of IRS-1. The sequencing was performed on thenoncoding strand (c.f. Table 3). Upper panel shows the wild typesequence whereas lower panel depicts the nucleotide sequence from anindividual who is heterozygous for a single base substitution in thecoding strand at nucleotide position 3494 as indicated by an arrow,(G→A) (c.f. Table 3). The base substitution in codon⁹⁷² caused asubstitution of glycine with arginine.

DETAILED DESCRIPTION OF THE INVENTION

In particular, the present invention relates to a DNA constructcomprising a DNA sequence encoding IRS-1 and containing a mutationgiving rise to at least one amino acid substitution in the IRS-1 proteinsequence. The mutation may for instance be located at a site where theamino acid substitution interferes with signal transduction throughIRS-1, as such a mutation is most likely to be involved in diseaseetiology. An example of such a DNA sequence is one containing a mutationof G to A in the first position of codon 972 of the IRS-1 gene.

This mutation leads to substitution of glycine by arginine in position972. The molecular mechanism by which the IRS-1 gene variant may lead todevelopment of NIDDM is not known at present. However, compared withglycine, arginine is a much larger molecule and has a polar side chainwith a positive charge and a high pKa of 12.5. In IRS-1, codon 972 islocated between two YMXM motifs. Although the changes which the glycinefor arginine substitution may cause in the tertiary structure of IRS-1are not known at present, it is assumed that the steric andelectrostatic changes of the IRS-1 mutant interfere with the normalinteraction between insulin and IGF-1 mediated phosphorylation ofneighbouring YMXM motifs of IRS-1 and signal transmitting proteins withsrc homology 2 domains. Alternatively, the mutation may be a markerassociated with another mutation in this or another gene, which othermutation is the one actually involved in disease etiology. This may alsobe the case with the silent mutations found in the third position ofcodon 805 (A to G) and in the third position of codon 894 (G to C).

Clinical investigations have shown that NIDDM patients as a whole haveinsulin resistant glucose disposal to peripheral tissues when comparedwith glucose-tolerant control subjects. However, glycine⁹⁷² -mutationcarriers with NIDDM did not differ in their degree of insulin resistancewhen compared with mutation-negative diabetic patients. Furthermore, thesensitivity of peripheral tissues to insulin was measured in 2 of 3mutation-positive nondiabetics who turned out to have values of insulinsensitivity within the normal range. In contrast, all mutation carriershad remarkably low fasting plasma concentrations of insulin andC-peptide when compared with matched noncarriers. The combination ofcomparable insulin sensitivity of peripheral tissues and low basallevels of circulating β-cell secretory products may indicate pancreaticβ-cell dysfunction.

In a preferred embodiment, the DNA construct of the invention comprisesthe DNA sequence shown in the Sequence Listing as SEQ ID NO:1, or afragment of said DNA sequence including the mutation of G to A atnucleotide 3494 of SEQ ID NO:1.

The length of the DNA construct may vary widely depending on theintended use. For use as an oligonucleotide probe for hybridisationpurposes, the DNA fragment may be as short as 17 nucleotides. Forexpression in a living system as defined above, the DNA construct willtypically comprise the full-length DNA sequence encoding IRS-1.

The DNA construct of the invention comprising the mutation in the DNAsequence encoding IRS-1 may suitably be of genomic or cDNA origin, forinstance obtained by preparing a genomic or cDNA library and screeningfor DNA sequences coding for all or part of the IRS-1 by hybridizationusing synthetic oligonucleotide probes in accordance with standardtechniques (cf. Sambrook et al., Molecular Cloning: A Laboratory Manual,2nd Ed., Cold Spring Harbor, 1989). The probes used should be specificfor the mutation. Alternatively, the DNA sequence encoding wild-typeIRS-1 may be modified by site-directed mutagenesis using syntheticoligonucleotides containing the mutation for homologous recombination inaccordance with well-known procedures. The DNA sequence may also beprepared by polymerase chain reaction using specific primers, forinstance as described in U.S. Pat. No. 4,683,202, or Saiki et al.,Science 239, 1988, pp. 487-491.

The DNA construct of the invention may also be prepared synthetically byestablished standard methods, e.g. the phosphoamidite method describedby Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859-1869, orthe method described by Matthes et al., EMBO Journal 3 (1984), 801-805.According to the phosphoamidite method, oligonucleotides aresynthesized, e.g. in an automatic DNA synthesizer, purified, annealedand ligated. This procedure may preferably be used to prepare fragmentsof the IRS-1 encoding DNA sequence.

The recombinant expression vector into which the DNA construct isinserted may be any vector which may conveniently be subjected torecombinant DNA procedures. The choice of vector will often depend onthe host cell into which it is to be introduced. Thus, the vector may bean autonomously replicating vector, i.e. a vector which exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g. a plasmid. Alternatively, the vector maybe one which, when introduced into a host cell, is integrated into thehost cell genome and replicated together with the chromosome(s) intowhich it has been integrated (e.g. a viral vector).

In the vector, the mutant DNA sequence encoding IRS-1 should be operablyconnected to a suitable promoter sequence. The promoter may be any DNAsequence which shows transcriptional activity in the host cell of choiceand may be derived from genes encoding proteins either homologous orheterologous to the host cell. Examples of suitable promoters fordirecting the transcription of the mutant DNA encoding IRS-1 inmammalian cells are the SV40 promoter (Subramani et al., Mol. Cell Biol.1 (1981), 854-864), the MT-1 (metallothionein gene) promoter (Palmiteret al., Science 222 (1983), 809-814) or the adenovirus 2 major latepromoter.

The mutant DNA sequence encoding IRS-1 may also be operably connected toa suitable terminator, such as the human growth hormone terminator(Palmiter et al., op. cit.). The vector may further comprise elementssuch as polyadenylation signals (e.g. from SV40 or the adenovirus 5 Elbregion), transcriptional enhancer sequences (e.g. the SV40 enhancer) andtranslational enhancer sequences (e.g. the ones encoding adenovirus VARNAs).

The recombinant expression vector may further comprise a DNA sequenceenabling the vector to replicate in the host cell in question. Anexample of such a sequence is the SV40 origin of replication. The vectormay also comprise a selectable marker, e.g. a gene the product of whichcomplements a defect in the host cell, such as the gene coding fordihydrofolate reductase (DHFR) or one which confers resistance to adrug, e.g. neomycin, hygromycin or methotrexate.

The procedures used to ligate the DNA sequences coding for IRS-1, thepromoter and the terminator, respectively, and to insert them intosuitable vectors containing the information necessary for replication,are well known to persons skilled in the art (cf., for instance,Sambrook et al., op.cit.).

In a further aspect, the present invention relates to a variant of IRS-1containing at least one amino acid substitution, in particular a variantcontaining at least one amino acid substitution at a site where thesubstitution interferes with signal transduction through IRS-1, or afragment thereof including said substitution. An example of such avariant is one in which glycine⁹⁷² is substituted by arginine, or afragment thereof containing said substitution, e.g. the variant whichhas the amino acid sequence shown in the Sequence Listing as SEQ IDNO:2, or a fragment thereof containing Arg⁹⁷².

The living system into which the DNA construct of the invention isintroduced may be a cell which is capable of producing IRS-1 and whichhas the appropriate signal transduction pathways. The cell is preferablya eukaryotic cell, such as a vertebrate cell, e.g. a Xenopus laevisoocyte or mammalian cell, in particular a mammalian cell. Examples ofsuitable mammalian cell lines are the COS (ATCC CRL 1650), BHK (ATCC CRL1632, ATCC CCL 10), CHL (ATCC CCL39) or CHO (ATCC CCL 61) cell lines.Methods of transfecting mammalian cells and expressing DNA sequencesintroduced in the cells are described in e.g. Kaufman and Sharp, J. Mol.Biol. 159 (1982), 601-621; Southern and Berg, J. Mol. Appl. Genet. 1(1982), 327-341; Loyter et al., Proc. Natl. Acad. Sci. USA 79 (1982),422-426; Wigler et al., Cell 14 (1978), 725; Corsaro and Pearson,Somatic Cell Genetics 7 (1981), 603, Graham and van der Eb, Virology 52(1973), 456; and Neumann et al., EMBO J. 1 (1982), 841-845.

The mutant DNA sequence encoding IRS-1 may then be expressed byculturing a cell as described above in a suitable nutrient medium underconditions which are conducive to the expression of the IRS-1-coding DNAsequence. The medium used to culture the cells may be any conventionalmedium suitable for growing mammalian cells, such as a serum-containingor serum-free medium containing appropriate supplements. Suitable mediaare available from commercial suppliers or may be prepared according topublished recipes (e.g. in catalogues of the American Type CultureCollection).

The living system according to the invention may also comprise atransgenic animal. A transgenic animal is one in whose genome aheterologous DNA sequence has been introduced. In particular, thetransgenic animal is a transgenic non-human mammal, mammals beinggenerally provided with appropriate signal transduction pathways. Themammal may conveniently be a rodent such as a rat or mouse. The mutantDNA sequence encoding IRS-1 may be introduced into the transgenic animalby any one of the methods previously described for this purpose.Briefly, the DNA sequence to be introduced may be injected into afertilised ovum or cell of an embryo which is subsequently implantedinto a female mammal by standard methods, resulting in a transgenicmammal whose germ cells and/or somatic cells contain the mutant DNAsequence. For a more detailed description of a method of producingtransgenic mammals, vide B. Hogan et al., Manipulating the Mouse Embryo,Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. The mutantDNA sequence may also be introduced into the animal by transfection offertilised ova with a retrovirus containing the DNA sequence, cf. R.Jaenisch, Proc. Natl. Acad. Sci. USA 73, 1976, pp. 1260-1264. A furthermethod of preparing transgenic animals is described in Gordon andRuddle, Methods Enzymol. 101, 1983, pp. 411-432.

In one embodiment of the present method of detecting the presence of amutation in the IRS-1 gene, a biological sample is obtained from asubject, DNA (in particular genomic DNA) is isolated from the sample anddigested with a restriction endonuclease which cleaves DNA at the siteof the mutation, and cleavage of the DNA within the gene encoding IRS-1at this site is determined. After digestion, the resulting DNA fragmentsmay be subjected to electrophoresis on an agarose gel. DNA from the gelmay then be blotted onto a nitrocellulose filter and hybridised with aradiolabelled probe. The probe may conveniently contain a DNA fragmentof the IRS-1 gene spanning the mutation (substantially according to themethod of E. M. Southern, J. Mol. Biol. 98, 1975, pp. 503, e.g. asdescribed by B. J. Conner et al., Proc. Natl. Acad. Sci. USA 80, 1983,pp. 278-282).

In a variant of this embodiment, the DNA isolated from the sample may beamplified prior to digestion with the restriction endonuclease.Amplification may suitably be performed by polymerase chain reaction(PCR) using oligonucleotide primers based on the appropriate sequence ofIRS-1 spanning the site(s) of mutation, essentially as described bySaiki et al., Science 230, 1985, pp. 1350-1354. After amplification, theamplified DNA may be digested with the appropriate restrictionendonuclease and subjected to agarose gel electrophoresis. Therestriction pattern obtained may be analysed, e.g. by staining withethidium bromide and visualising bands in the gel by means of UV light.As a control, wild-type DNA encoding IRS-1 (i.e. not containing themutation) may be subjected to the same procedure, and the restrictionpatterns may be compared.

In the method of the invention, the sample is preferably analysed for amutation located at a site where amino acid substitution interferes withsignal transduction through IRS-1. A specific example of such a mutationis the mutation of G to A in the first position of codon 972 of the geneencoding IRS-1. This mutation results in a new restriction endonucleasecleavage site

5'-C C A/T G G-3' (SEQ ID NO:3)

3'-G G T/A C C-5' (SEQ ID NO:3) in the mutant DNA sequence coding forIRS-1.

An example of a suitable restriction endonuclease is BstN1.

A further embodiment of the method of the invention is an adaptation ofthe method described by U. Landegren et al., Science 241, 1988, pp.1077-1080, which involves the ligation of adjacent oligonucleotides on acomplementary target DNA molecule. Ligation will occur at the junctionof the two oligonucleotides if the nucleotides are correctly basepaired.

In a still further embodiment of the present method, the DNA isolatedfrom the sample may be amplified using oligonucleotide primerscorresponding to segments of the gene coding for IRS-1. The amplifiedDNA may then be analysed by hybridisation with a labelledoligonucleotide probe comprising a DNA sequence corresponding to atleast part of the gene encoding IRS-1 and containing a mutation of atleast one nucleotide, which mutation corresponds to the mutation thepresence of which in the gene encoding IRS-1 is to be detected. As acontrol, the amplified DNA may furthermore be hybridised with a labelledoligonucleotide probe comprising a DNA sequence corresponding to atleast part of the wild-type gene encoding IRS-1. This procedure is, forinstance, described by DiLella et al., Lancet 1, 1988, pp. 497-499.Another PCR-based method which may be used in the present invention isthe allele-specific PCR method described by R. Saiki et al., Nature 324,1986, pp. 163-166, or D. Y. Wu et al., Proc. Natl. Acad. Sci. USA 86,1989, pp. 2757-2760, which uses primers specific for the mutation in theIRS-1 gene.

Other methods of detecting mutations in DNA are reviewed in U.

Landegren, GATA 9, 1992, pp. 3-8. A currently preferred method ofdetecting mutations is by single stranded conformation polymorphism(SSCP) analysis substantially as described by Orita et al., Proc. Natl.Acad. Sci. USA 86, 1989, pp. 2766-2770, or Orita et al., Genomics 5,1989, pp. 874-879.

The label substance with which the probe is labelled is preferablyselected from the group consisting of enzymes, coloured or fluorescentsubstances, or radioactive isotopes.

Examples of enzymes useful as label substances are peroxidases (such ashorseradish peroxidase), phosphatases (such as acid or alkalinephosphatase), β-galactosidase, urease, glucose oxidase, carbonicanhydrase, acetylcholinesterase, glucoamylase, lysozyme, malatedehydrogenase, glucose-6-phosphate dehydrogenase, β-glucosidase,proteases, pyruvate decarboxylase, esterases, luciferase, etc.

Enzymes are not in themselves detectable but must be combined with asubstrate to catalyse a reaction the end product of which is detectable.Examples of substrates which may be employed in the method according tothe invention include hydrogen peroxide/tetramethylbenzidine orchloronaphthole or o-phenylenediamine or 3-(p-hydroxyphenyl) propionicacid or luminol, indoxyl phosphate, p-nitrophenylphosphate, nitrophenylgalactose, 4-methyl umbelliferyl-D-galactopyranoside, or luciferin.

Alternatively, the label substance may comprise coloured or fluorescentsubstances, including gold particles, coloured or fluorescent latexparticles, dye particles, fluorescein, phycoerythrin or phycocyanin.

In a particularly favoured embodiment, the probe is labelled with aradioactive isotope. Radioactive isotopes which may be used for thepresent purpose may be selected from I-125, I-131, In-111, H-3, P-32,C-14 or S-35. The radioactivity emitted by these isotopes may bemeasured in a beta- or gamma-counter or by a scintillation camera in amanner known per se.

For use in the present method, the invention further relates to a testkit for detecting the presence of a mutation in the gene encoding IRS-1,the kit comprising

(a) a restriction endonuclease which cleaves DNA at the site of themutation,

(b) a first DNA sequence corresponding to at least part of the wild-typegene encoding IRS-1, and/or

(c) a second DNA sequence corresponding to at least part of the geneencoding IRS-1 and containing a mutation of at least one nucleotide,which mutation corresponds to the mutation the presence of which in thegene encoding IRS-1 is to be detected.

The first DNA sequence may, for instance, be obtained from genomic DNAor cDNA encoding IRS-1 obtained from a healthy subject. The second DNAsequence may conveniently be a DNA construct according to the invention.

For use in the present method, the invention further relates to a testkit for detecting the presence of a mutation in the gene encoding IRS-1,the kit comprising

(a) means for amplifying DNA, and

(b) a labelled oligonucleotide probe comprising a DNA sequencecorresponding to at least part of the gene encoding IRS-1 and containinga mutation of at least one nucleotide, which mutation corresponds to themutation the presence of which in the gene encoding IRS-1 is to bedetected.

Appropriate means for amplifying DNA (typically genomic DNA isolatedfrom the biological sample) include, for instance, oligonucleotideprimers, appropriate buffers and a thermostable DNA polymerase.

The invention is further illustrated in the following example which isnot intended in any way to limit the scope of the invention as claimed.

EXAMPLES Example 1

Subjects

A total of 86 NIDDM patients and 76 control subjects were included inthe protocol. All study participants were unrelated Danish Caucasiansand their clinical characteristics are given in Table 1 and 2. Thecontrol subjects had normal fasting plasma glucose levels and no familyhistory of known diabetes. Patients with NIDDM, as defined by theNational Diabetes Data Group (18), were recruited consecutively andunselected from the outpatient clinic at Steno Diabetes Center. AllNIDDM patients were treated with diet, oral hypoglycemic drugs, or both.None of the participants in the study suffered from liver or kidneydiseases as evaluated by clinical and standard laboratory examinations,and no subject was taking any other medication known to influencepancreatic β-cell function or energy metabolism. Before participation,the purpose and risks of the study were carefully explained to allvolunteers and their informed consent was obtained. The protocol wasapproved by the local Ethical Committee of Copenhagen and was inaccordance with the Helsinki declaration.

Euglycemic, Hyperinsulinemic Clamp

NIDDM patients and 19 glucose-tolerant healthy control subjects wereexamined using a euglycemic, hyperinsulinemic clamp. The experimentswere undertaken in the fasting state between 08.00 and 15.00 after a 10h overnight fast. Each clamp comprised a 2 h basal period followed by a4 h hyperinsulinemic glucose clamp. Details of the clamp technique havebeen described previously (19). To assess total peripheral glucoseuptake, (3-³ H) glucose was infused throughout the study period. In thecontrol subjects, (3-³ H) glucose was administered as a primed (25-μCi)continuous (0.25-μCi/min) infusion, whereas in the NIDDM subjects, thepriming was increased in proportion to the increase in fasting plasmaglucose concentration; the continuous infusion of labeled glucose wasthe same as in the control subjects (0.25 μCi/min). The clamp wasperformed by continuous infusion of 2 mU insulin×kg⁻¹ ×min⁻¹ (Actrapid,Novo Nordisk, Denmark), and euglycemia was maintained by a variableinfusion of 20% glucose at a rate determined by the measurement ofplasma glucose levels at 5 to 10 min invervals. Total glucose disposalrate was calculated from the plasma concentrations of (3-³ H) glucoseand plasma glucose with Steele's non-steady state equations (20). Inthese calculations, the distribution volume of glucose was taken as 200ml/kg body weight and the pool fraction as 0.65. At the highest steadystate plasma insulin level, where the hepatic glucose production ispresumed to be nil, glucose infusion rates were used to calculateglucose disposal. Total peripheral glucose uptake was corrected forurinary glucose loss.

Preparation and Amplification of Genomic DNA

Genomic DNA was isolated from human leucocyte nuclei isolated from wholeblood by protein kinase K digestion followed by phenol extraction on aApplied Biosystems 341 Nucleic Acid Purification System. A primary 50 μlPCR reaction was carried out with 0,3 μg of genomic DNA. The assayconditions were: 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl₂, 0.1%Triton X-100, 0.2 mM dNTP's, 0.2 μM of each oligonucleotide primer and1.25 u Taq DNA polymerase (Promega, Madison, Wis.). Specificoligonucleotide primers for the human IRS-1 gene (4), 20-25 mers and 2G's or C's at the 3' end, were synthezised on an Applied Biosystems 394DNA/RNA syntheziser (Applied Biosystems Inc., Foster City, Calif.) andeluted on a NAB-10 column (Pharmacia P-L Biochemicals Inc, Milwaukee,Wis.) and used without further purification.

The nucleotide sequences of the DNA primers used for the polymerasechain reactions were as follows:

    ______________________________________                                         1.  553   5'GCTCAGCGTTGGTGGTGGCGGTGG3'                                                                         577 (SEQ ID                                                                   NO: 4)                                       2.  783   3'CGACGAAGTTGTAGTTGTTCGCCCG5'                                                                        807 (SEQ ID                                                                   NO: 5)                                       3.  727   5'GAAGTGGCGGCACAAGTCGAGCGC3'                                                                         756 (SEQ ID                                                                   NO: 6)                                       4.  999   3'CGAGGCCGGAACCACTCCGACC5'                                                                           1020 (SEQ ID                                                                  NO: 7)                                       5.  924   5'ACCGTGCTAAGGGCCACCACGACG3'                                                                         947 (SEQ ID                                                                   NO: 8)                                       6. 1180   3'CCTCCGTCGCCGGCACCACGA5'                                                                            1200 (SEQ ID                                                                  NO: 9)                                       7. 1128   5'TCTACCGCCTTTGCCTGACCAGC3'                                                                          1150 (SEQ ID                                                                  NO: 10)                                      8. 1392   3'CGGTCAGGAGCAGGTTGACG5'                                                                             1411 (SEQ ID                                                                  NO: 11)                                      9. 1337   5'ACCATCCTGGAGGCCATGCGG3'                                                                            1357 (SEQ ID                                                                  NO: 12)                                     10. 1598   3'GGTCGGAGCCACCTGCCGTCGGGA5'                                                                         1621 (SEQ ID                                                                  NO: 13)                                     11. 1545   5'CAGGCTCCTTCCGTGTCCGCG3'                                                                            1565 (SEQ ID                                                                  NO: 14)                                     12. 1807   3'GGGTGCCGCTAGATCACGAAG5'                                                                            1827 (SEQ ID                                                                  NO: 15)                                     13. 1757   5'CTGTCGTCCAGTAGCACCAGTGG3'                                                                          1779 (SEQ ID                                                                  NO: 16)                                     14. 2007   3'GGGACTGGCGGGGGTTGCCAG5'                                                                            2037 (SEQ ID                                                                  NO: 17)                                     15. 1953   5'GCGGTGAGGAGGAGCTAAGC3'                                                                             1932 (SEQ ID                                                                  NO: 18)                                     16. 2200   3'GGGCAGGGTCAGGAGTCACCG5'                                                                            2230 (SEQ ID                                                                  NO: 19)                                     17. 2151   5'AGAGAACTCACTCGGCAGGC3'                                                                             2170 (SEQ ID                                                                  NO: 20)                                     18. 2401   3'GGTGTGCCTACTACCGATGTACGG5'                                                                         2424 (SEQ ID                                                                  NO: 21)                                     19. 2352   5'ACCCCTTGGAGCGTCGGGGG3'                                                                             2371 (SEQ ID                                                                  NO: 22)                                     20. 2600   3'GGACTGAATCCTCCACCGGGGTCG5'                                                                         2623 (SEQ ID                                                                  NO: 23)                                     21. 2546   5'CAGAGAGTGGACCCCAATGG3'                                                                             2566 (SEQ ID                                                                  NO: 24)                                     22. 2800   3'CCTGAGGTTGTGGTCGTCGG5'                                                                             2819 (SEQ ID                                                                  NO: 25)                                     23. 2712   5'TCTTGCCTCACCCCAAACCC3'                                                                             3150 (SEQ ID                                                                  NO: 26)                                     24. 2964   3'CGAGACCAGCGGAAGAGATA5'                                                                             2983 (SEQ ID                                                                  NO: 27)                                     25. 2918   5'GAGCCGGAGGAGGGTGCCCG3'                                                                             2938 (SEQ ID                                                                  NO: 28)                                     26. 3180   3'GGTTCCGGTCGTGGAATGGA5'                                                                             3199 (SEQ ID                                                                  NO: 29)                                     27. 3131   5'CAGACCAATAGCCGCCTGGC3'                                                                             3150 (SEQ ID                                                                  NO: 30)                                     28. 3392   3'CCGTGACTCCTCATGTACTT5'                                                                             3411 (SEQ ID                                                                  NO: 31)                                     29. 3339   5'CTTCTGTCAGGTGTCCATCC3'                                                                             3358 (SEQ ID                                                                  NO: 32)                                     30. 3582   3'CGATGCACCTGTGGAGCGGT5'                                                                             3601 (SEQ ID                                                                  NO: 33)                                     31. 3532   5'GGGCAGTGCCCAGCAGCCGG3'                                                                             3541 (SEQ ID                                                                  NO: 34)                                     32. 3743   3'CGACGGGTGAGCAGGGACGACC5'                                                                           3764 (SEQ ID                                                                  NO: 35)                                     33. 3687   5'CCTCAGCAGCCTCTGCTTCC3'                                                                             3712 (SEQ ID                                                                  NO: 36)                                     34. 3950   3'CGTCGTCATCCCCCGCCACC5'                                                                             3969 (SEQ ID                                                                  NO: 37)                                     35. 3900   5'CCACACCCAGTGCCACCCGG3'                                                                             3919 (SEQ ID                                                                  NO: 38)                                     36. 4141   3'CCTGAAGTTTGTCACGGGAG5'                                                                             4160 (SEQ ID                                                                  NO: 39)                                     37. 4067   5'GAGCCAGCCAAACTGTGTGG3'                                                                             4086 (SEQ ID                                                                  NO: 40)                                     38. 4320   3'CCTGTAGTGTCGTCCAGCAA5'                                                                             4339 (SEQ ID                                                                  NO: 41)                                     ______________________________________                                    

The mixture was overlaid with 40 μl of mineral oil and after initialdenaturation at 95° C. for 3 min, the samples were subjected to 35cycles of amplification: annealing at 60° C. for 1 min, extension at 72°C. for 2 min and denaturation at 94° C. for 1 min. In these primary PCRreactions, fragments of 800-900 bp, were amplified. A secondary PCRreaction was performed with 1 μl of a 1:10 diluted primary PCR productas template. The amplification conditions were as described above,except that (α-³² P)dCTP (Amersham International, Buckinghamshire, UK)was added. Each fragment amplified during secondary PCR had a size of250-285 bp.

Single Stranded Conformation Polymorphism (SSCP) Analysis

In the primary mutation screening, SSCP analysis (according to themethod of Orita et al., Genomics 5, 1989, pp. 874-879) of the entirecoding region of IRS-1 was performed on genomic DNA from 19 insulinresistant NIDDM patients and 5 control subjects (Table 1). In all cases,2 μl of a secondary PCR product was combined with 8 μl of a sequencingstop solution. One μl was loaded unheated to identify double-strandedproducts and after heating at 94° C. for 5 min, 2 μl was loaded to thesame wells on a 38×31×0,03 cm 5% polyacrylamid gel (49:1,acrylamid:bisacrylamid) in 90 mM Tris-borate, 2,5 mM EDTA, with 1% or 5%glycerol, respectively. In each patient SSCP analysis was undertaken at2 different experimental conditions: electrophoresis was carried outwith 1% glycerol at 35 W constant power for 4-5 h at 4° C. by placinggel, buffer and electrophoresis apparatus at 4° C. overnight, and with5% glycerol at 65 W constant power for 2-3 hours at 25° C. (21). Thegels were transferred to 3 MM filter paper, covered with a plastic wrapand autoradiographed at -80° C. with intensifying screens for 2-10 h.Gels contained 2 lanes with a known plasmid mutation and thecorresponding wild type fragment as positive controls. In our laboratorythe ability to detect known plasmid mutations (single basesubstitutions, small insertions, or small deletions) is 80-90% (22).

Synthesis of Single-Stranded DNA and Direct Nucleotide Sequencing

Single stranded DNA for sequencing was generated using biotinylatedoligonucleotide primers and streptavidin-coated magnetic beads(available from Dynal AS, Oslo, Norway). Products were precipitated withammoniumacetate and isopropanol and resuspended in appropiate volumes ofwater. Sequencing using the AutoRead Sequencing Kit (Pharmacia, Uppsala,Sweden) and Fluore-dATP was analysed on an automated laser fluorescenceDNA sequencer (Pharmacia, Uppsala, Sweden).

Direct Restriction Enzyme Digestion of PCR Products

Restriction enzyme digestion was carried out in 15 μl reactions,containing 10 μl precipitated secondary PCR product, 1,5 μl 10× NEbuffer 2, 100 μg/ml bovine serum albumin and 10 u of the restrictionenzyme BstN 1 (New England BioLabs Inc., Beverly, Mass.). The fragmentswere analyzed on a 4.5% high solution agarose gel and visualized afterstaining with ethidium bromide.

Synthesis of cDNA

Percutaneous muscle biopsies (˜500 mg) were obtained under localanesthesia from the vastus lateralis muscle of the thigh. Muscle sampleswere blotted to remove blood and connective and adipose tissue and werewithin 30 sec frozen in liquid N₂ and stored at -80° C. until assayed.Total RNA was isolated from muscle biopsies as described (23). cDNA wassynthesized in 25 μl volumes containing 1 μg of total RNA, 0.2 mMdeoxynucleoside triphosphates, 40 u RNasin (Promega, Madison, Wis.),0.625 μg oligo (dT)₁₈, 400 u Moloney Murine Leukaemia Virus ReverseTranscriptase (Life Technologies Inc., Grand Island, N.Y.), 50 mMTris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂ and 10 mM DTT. The reactionswere performed at 37° C. for 1 hour, followed by enzyme inactivation byincubation for 10 min at 95° C. SSCP was performed on cDNA as describedfor genomic DNA.

Other Assays

Plasma for analysis of chemical quantities was drawn from all studyparticipants in the morning after 10 h of overnight fast. Plasma glucosewas measured in duplicate by a hexokinase method. Tritiated glucose inplasma was determined as described (24). HbA_(1C) was measured using aHPLC method (normal range 4.1-6.4%). Plasma insulin and C-peptide levelswere analyzed by radioimmuno-assays (25,26).

Statistical Analysis

Data in text and tables are given as means±SE. For comparisonsMann-Whitney's test for unpaired data was applied.

Primary Screening for Mutations in the IRS-1 Gene

In 19 overlapping fragments, each consisting of 250-285 bp, the entirecoding region of the IRS-1 gene was analyzed using genomic DNA from 19NIDDM patients and 5 control subjects. Compared with matched controlsubjects the NIDDM patients were as a group hyperinsulinemic (p<0.02)and were characterized by impaired insulin stimulated glucose disposalto peripheral tissues (p<0.0001) (Table 1). SSCP scanning showed 3different aberrant migration profiles. Subsequent nucleotide sequencingrevealed one polymorphism at codon 805 (GCA→GCG (alanine)). Four of 19NIDDM patients were heterozygous for this single base substitution whichdid not predict any change in the amino acid sequence. Another silentmutation was detected at codon 894 (CCG→CCC (proline)). Only one of 19NIDDM patients was heterozygous for this nucleotide base variation.However, at codon 972 SSCP analysis showed that three of 19 NIDDMpatients were heterozygous for a mutation in which glycine (GGG) wassubstituted for arginine (AGG) (FIGS. 1 and 2). The mutation wasconfirmed by sequencing of both DNA strands and by direct enzymaticdigestion of PCR products with the restriction enzyme Bst N1, for whicha restriction site was created by the nucleotide substitution (data notshown). None of the 5 control subjects, who primarily were SSCP scanned,showed evidence of polymorphisms. The glycine 972 mutation was locatedbetween 2 Tyr-Met-X-Met motifs in the IRS-1 gene (Table 3).Subsequently, the codon 972 mutation, primarily identified on genomicDNA isolated from blood cells, was verified by studies of cDNA,synthesized from total RNA which was isolated from skeletal musclebiopsies.

Secondary Screening for Glycine⁹⁷² Mutation

Using SSCP molecular scanning and specific enzymatic digestion ofprimary PCR products further 67 NIDDM patients (Table 2) and 76 healthycontrol subjects were examined for the occurrence of the glycine⁹⁷²mutation. Seven of the 67 NIDDM patients were heterozygous for themutation. Moreover, 3 of 76 healthy volunteers were also heterozygouscarriers of the codon⁹⁷² mutation. All 3 control subjects who werepositive for the mutation had a normal glucose response to an oralglucose challenge (data not shown).

Clinical Characterization of Individuals Carrying the Glycine⁹⁷²Mutation

Table 1 shows the results obtained from the clinical investigations ofthe 3 glucose-tolerant controls who were positive for the glycine⁹⁷²mutation. Insulin-glucose clamp was performed in 2 of the 3 mutationcarriers and the glucose disposal rates of these individuals were withinthe range of the 19 mutation-negative control subjects. However,interestingly also glucose- tolerant mutation carriers werecharacterized by relatively low values of fasting plasma insulin(decreased by 33-46%) and C-peptide (decreased by 25-40%), respectively,when compared with mean values obtained in mutation-negative healthycontrols.

Table 2 gives a summary of the phenotypical characteristics of the 10NIDDM patients who are heterozygous carriers of the glycine⁹⁷² mutationin the IRS-1 gene when compared to 76 NIDDM patients who are negativefor the same mutation. No significant differences were shown in age,known diabetes duration, body mass index, fasting levels of plasmaglucose, HbA_(1C) and basal or insulin stimulated glucose disposalrates. However, fasting plasma levels of insulin and C-peptide werereduced by 44% (p <0.05) and 37% (p<0.02), respectively, in diabeticswho were positive for the glycine⁹⁷² mutation when compared withmutation-negative diabetic subjects.

Example 2

A random sample of 383 unrelated healthy young Caucasians (15-32 yearsof age) was studied to determine whether the glycine⁹⁷² mutation confersinsulin resistance. All individuals had their insulin sensitivityestimated using Bergman's minimal model (intravenous injections ofglucose combined with tolbutamide), and the occurrence of the glycine⁹⁷²mutation was determined by means of SSCP scanning (as described inExample 1) and restriction enzyme analysis of genomic DNA (as describedin Example 1). 35 subjects were found to carry the mutation.

Those carriers of the glycine⁹⁷² mutation who had a body mass index(BMI) of more than 25 kg/m² (n=10, BMI=28.4±0.9 kg/m²) (mean±standarderror) had a two-fold lower insulin sensitivity and a significantlyhigher fasting serum level of C-peptide than the non-carriers with thesame BMI (n=99, BMI=28.1±0.3 kg/m²). In contrast, there was nodifference in the measured variables between the carriers andnon-carriers of the mutation in subjects with a BMI of less than 25kg/m². In multivariate analysis adjusting for differences in VO₂ max,BMI, gender and age, the presence of the glycine⁹⁷² mutation within thegroup of obese subjects was negatively associated with insulinsensitivity (p<0.04) and positively associated with fasting serumC-peptide (p<0.09), fasting serum triglyceride (p<0.02) and serum totalcholesterol (p<0.03).

The results of the study show that in young obese subjects, theglycine⁹⁷² mutation is associated with whole-body insulin resistance anddyslipidemia.

REFERENCES

1. Hitman GA, McCarthy MI. Genetics of non-insulin-dependent diabetesmellitus. Baillieres Clin Endocrinol Metab 1991; 5:455-476.

2. DeFronzo RA. The triumverate: β-cell, muscle, liver: a collisionresponsible for NIDDM. Diabetes 1988; 37:667-687.

3. Kahn CR, White MF. The insulin receptor and the molecular mechanismof insulin action. J Clin Invest 1988; 82:1151-1156.

4. Sun XJ, Rothenberg P, Kahn CR, et al. Structure of the insulinreceptor substrate IRS-1 defines a unique signal transduction protein.Nature (Lond ) 1991; 352:73-77.

5. Rothenberg P, Lane WS, Karasik A, Backer J, White MF, Kahn CR.Purification and partial sequence analysis of pp185, the major cellularsubstrate of the insulin receptor tyrosine kinase. J Biol Chem 1991;266:8302-8311.

6. Nishiyama M, Wands JR. Cloning and increased expression of an insulinreceptor substrate-1-like gene in human hepatocellular carcinoma.Biochem Biophys Res 1992; 183:280-285.

7. Backer JM, Schroeder GG, Kahn CR, et al. Insulin stimulation ofphosphatidylinositol 3-kinase activity maps to insulin receptor regionsrequired for endogenous substrate phosphorylation. J Biol Chem 1992;267:1367-1364.

8. Myers Jr MG, Backer JM, Sun XJ, et al. IRS-1 activatesphosphatidylinositol 3-kinase by associating with src homology 2 domainsof p85. Proc Natl Acad Sci USA 1992; 89:10350-10354.

9. Backer JM, Myers Jr MG, Shoelson SE, et al. Phosphatidylinositol3-kinase is activated by association with IRS-1 during insulinstimulation. The EMBO Journal 1992; 11: 3469-3479.

10. Yonezawa K, Ueda H, Hara K, et al. Insulin-dependent formation of acomplex containing an 85-kDa subunit of phosphatidylinositol 3-kinaseand tyrosine-phosphorylated insulin receptor substrate 1. J Biol Chem1992; 267:25958-25966.

11. Folli F, Saad MJA, Backer JM, Kahn CR. Insulin stimulation ofphosphatidylinositol 3-kinase activity and association with insulinreceptor substrate 1 in liver and muscle of the intact rat. J Biol Chem1992; 267:22171-22177.

12. Yonezawa K, Yokono K, Shii K, et al. In vitro association ofphosphatidylinositol 3-kinase activity with the activated insulinreceptor tyrosine kinase. J Biol Chem 1992; 267:440-446.

13. Hayashi H, Kamohara S, Nishioka Y, et al. Insulin treatmentstimulates the tyrosine phosphorylation of the α-type 85-kDa subunit ofphosphatidylinositol 3-kinase in vivo. J Biol Chem 1992;267:22575-22580.

14. Sun XJ, Miralpeix M, Myers Jr MG, et al. Expression and function ofIRS-1 in insulin signal transmission. J Biol Chem 1992; 267:22662-22672.

15. Shoelson SE, Chatterjee S, Chaudhuri M, White MF. YMXM motifs ofIRS-1 define substrate specificity of the insulin receptor kinase. ProcNatl Acad Sci USA 1992; 89:2027-2031.

16. McClain DA, Maegawa H, Scott Thies R, Olefsky JM. Dissection of thegrowth versus metabolic effects of insulin and insulin-like growthfactor-I in transfected cells expressing kinase-defective human insulinreceptors. J Biol Chem 1990; 265:1678-1682.

17. Taylor SI, Cama A, Accili D. Molecular genetics of insulin resistantdiabetes mellitus. J Clin Endocrinol Metab 1991; 73:1158-1163.

18. National Diabetes Data Group . Classification and diagnosis ofdiabetes mellitus and other categories of glucose intolerance. Diabetes1979; 28:1039-1057.

19. DeFronzo RA, Tobin JD, Andres R. Glucose clamp technique: a methodfor quantifying insulin secretion and resistance. Am J Physiol 1979;237:E214-E223.

20. Steele R. Influence of glucose loading and of injected insulin onhepatic glucose production. Ann NY Acad Sci 1959; 82:420-430.

21. Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T. Detections ofpolymorphisms of human DNA by gel electrophoresis as single-strandedconformation polymorphisms. Proc Natl Acad Sci USA 1989; 86:2766-2770.

22. Vestergaard H, Bj.o slashed.rb.ae butted.k C, Andersen PH, Bak JF,Pedersen 0. Impaired expression of glycogen synthase mRNA in skeletalmuscle of NIDDM patients. Diabetes 1991; 40:1740-1745.

23. Chomczynski P, Sachhi N. Single-step method of RNA isolation by acidguanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156-159.

24. Hother-Nielsen O, Schmitz O, Bak JF, Beck-Nielsen H. Enhancedhepatic insulin sensitivity, but peripheral insulin resistance inpatients with type I (insulin-dependent) diabetes. Diabetologia 1987;30:834-840.

25. Heding LG. Determination of total serum insulin (IRI) ininsulin-treated diabetic patients. Diabetologia 1972; 8:260-266.

26. Heding LG. Radioimmunological determination of human C-peptide inserum. Diabetologia 1975; 11:541-548.

27. White MF, Maron R, Kahn CR. Insulin rapidly stimulates tyrosinephosphorylation of a Mr 185,000 protein in intact cells. Nature (Lond )1985; 318:183-186.

28. White MF, Stegmann EW, Dull TJ, Ullrich A, Kahn CR. Characterizationof an endogenous substrate of the insulin receptor in cultured cells. JBiol Chem 1987; 262:9769-9777.

                                      TABLE 1                                     __________________________________________________________________________    Phenotypical characterization of 19 NIDDM patients who were examined with     the primary SSCP                                                              scanning of the IRS-1 gene: comparisons to 19 matched controls without        detected IRS-1                                                                mutations and 3 glucose-tolerant controls in whom glycine.sup.972 was         substituted with arginine                                                              Body Mass Plasma                                                                            Plasma                                                                            Plasma                                             N     Age                                                                              Index HbA.sub.1C                                                                        glucose                                                                           insulin                                                                           C-peptide                                                                          M-value                                                                           M-value                                   (F:M) (yr)                                                                             (kg/m.sup.2)                                                                        (%) (mM)                                                                              (pM)                                                                              (nM) (basal)                                                                           (insulin)                                 __________________________________________________________________________    NIDDM                                                                         Mean                                                                             6/13                                                                             54 29    7.9 10.4                                                                              95  0.92 88  330                                       ±SE                                                                               2  1    0.5 0.9 13  0.09  3   30                                       CONTROL (noncarriers of mutation)                                             Mean                                                                             7/12                                                                             50 28     5.4*                                                                              5.5*                                                                               60+                                                                              0.67§                                                                          77+                                                                              484*                                     ±SE                                                                               2  1    0.1 0.1  6  0.04  2   30                                       CONTROL (carriers of mutation)                                                No. 1                                                                            F  50 27    4.7 5.1 32  0.40 71  461                                       No. 2                                                                            M  64 23    5.6 6.0 40  0.51 78  389                                       No. 3                                                                            M  66 23    6.0 5.9 34  0.42 ND  ND                                        __________________________________________________________________________     Plasma levels of glucose, insulin and Cpeptide were measured in the           fasting state in the morning. Mvalue is the glucose disposal rate             (mg/m.sup.2 /min) in the fasting state (basal) in the morning and after 4     hours of euglycemic and hyperinsulinemic clamp (insulin) with steady stat     plasma insulin concentrations during the last 30 min of the clamp of 1165     ± 71 pM in NIDDM patients and 1034 ± 56 pM in controls who were         noncarriers of the glycine.sup.972 mutation. In the 3 glucosetolerant         mutation carriers the steady state plasma insulin levels were 786 pM          (subject no. 1) and 1008 pM (subject no. 2), respectively.                    ND = not determined.                                                          *P < 10.sup.-4 vs. NIDDM;                                                     +P < 0.02 vs. NIDDM;                                                          §P < 0.03 vs. NIDDM.                                                

                  TABLE 2                                                         ______________________________________                                        Characteristics of 10 NIDDM patients who are carriers                         of the glycine.sup.972  -> arginine mutation in the IRS-1 gene:               comparisons to NIDDM patients who are noncarriers of the mutation                                +Mutation  -Mutation                                       ______________________________________                                        N (F/M)            3/7        30/46                                           Age (yr)           52 ± 2  54 ± 1                                       Body Mass Index (kg/m.sup.2)                                                                     29 ± 2  30 ± 1                                       Known duration of diabetes (yr)                                                                  5 ± 1   4 ± 1                                        HbA.sub.1C  (%)    8.6 ± 0.6                                                                             8.0 ± 0.2                                    Plasma glucose (mM)                                                                              12.4 ± 1.1                                                                            11.7 ± 0.5                                   Plasma insulin (pM)                                                                              53 ± 10 94 ± 8*                                      Plasma C-peptide (nM)                                                                            0.49 ± 0.06                                                                           0.78 ± 0.05 +                                M-value (basal)    86 ± 5  91 ± 3                                       M-value (insulin)  286 ± 29                                                                              265 ± 16                                     ______________________________________                                         Value are means ± SE. Plasma concentrations of glucose, insulin and        Cpeptide were measured in the fasting state in the morning. Mvalue is the     glucose disposal rate (mg/m.sup.2 /min) in the fasting state (basal) in       the morning and after 4 hours of euglycemic and hyperinsulinemic clamp        (insulin) with steady state plasma insulin concentrations during the last     30 min of the clamp of 980 ± 57 pM in NIDDM patients who were carriers     of the glycine.sup.972  mutation and 1153 ± 34 pM in NIDDM patients wh     were noncarriers, respectively.                                               *P < 0.05; +P < 0.02.                                                    

                                      TABLE 3                                     __________________________________________________________________________    Partial nucloetide (3392-3562) and amino acid (938-994) sequence of the       published human                                                               insulin receptor substrate 1 (IRS-1). The glycine to arginine mutation is     located at codon                                                              972 (shown in bold). Two YMXM motifs which are putative recognition sites     for insulin                                                                   signal transmission proteins carrying src homology-2 domains are              underlined.                                                                   __________________________________________________________________________     ##STR1##                                                                     aa938GlyThrGluGluTyrMetLysMetAspLeuGlyProGlyArgArgAlaAlaTrpGln                 ##STR2##                                                                      ##STR3##                                                                     ATTTGCAGGCCTACCCGGGCAGTGCCCAGCAGCCGGGGTGACTACATGACCATGCAG-3'                  3562                                                                          TAAACGTCCGGATGGGCCCGTCACGGGTCGTCGGCCCCACTGATGTACTGGTACGTC-5'                  IleCysArgProThrArgAlaValProSerSerArgGlyAspTyrMetThrMetGln (SEQ ID NOS: 1      and 2) 994                                                                    __________________________________________________________________________

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 41                                                 (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6152 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 581..4309                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      TGGTATTTGGGCGGCTGGTGGCGGCGGGGACTGTTGGAGGGTGGGAGGAGGCGAAGGAGG60                AGGGAGAACCCCGTGCAACGTTGGGACTTGGCAACCCGCCTCCCCCTGCCCAAGGATATT120               TAATTTGCCTCGGGAATCGCTGCTTCCAGAGGGGAACTCAGGAGGGAAGGGGGCGCGCGC180               TCCTGGAGGGGCACCGCAGGGACCCCCGACTGTCGCCTCCCTGTGCCGGACTCCAGCCGG240               GGCGACGAGAGATGCATCTTCGCTCCTTCCTGGTGGCGGCGGCGGCTGAGAGGAGACTTG300               GCTCTCGGAGGATCGGGGCTGCCCTCACCCCGGACGCACTGCCTCCCCGCCGGGCGTGAA360               GCGCCCGAAAACTCCGGTCGGGCTCTCTCCTGGGCTCAGCAGCTGCGTCCTCCTTCAGCT420               GCCCCTCCCCGCGCGGGGGGCGGCGTGGATTTCAGAGTCGGGGTTTCTGCTGCCTCCAGC480               CCTGTTTGCATGTGCCGGGCCGCGGCGAGGAGCCTCCGCCCCCCACCCGGTTGTTTTTCG540               GAGCCTCCCTCTGCTCAGCGTTGGTGGTGGCGGTGGCAGCATGGCGAGCCCTCCG595                    MetAlaSerProPro                                                               15                                                                            GAGAGCGATGGCTTCTCGGACGTGCGCAAGGTGGGCTACCTGCGCAAA643                           GluSerAspGlyPheSerAspValArgLysValGlyTyrLeuArgLys                              101520                                                                        CCCAAGAGCATGCACAAACGCTTCTTCGTACTGCGCGCGGCCAGCGAG691                           ProLysSerMetHisLysArgPhePheValLeuArgAlaAlaSerGlu                              253035                                                                        GCTGGGGGCCCGGCGCGCCTCGAGTACTACGAGAACGAGAAGAAGTGG739                           AlaGlyGlyProAlaArgLeuGluTyrTyrGluAsnGluLysLysTrp                              404550                                                                        CGGCACAAGTCGAGCGCCCCCAAACGCTCGATCCCCCTTGAGAGCTGC787                           ArgHisLysSerSerAlaProLysArgSerIleProLeuGluSerCys                              556065                                                                        TTCAACATCAACAAGCGGGCTGACTCCAAGAACAAGCACCTGGTGGCT835                           PheAsnIleAsnLysArgAlaAspSerLysAsnLysHisLeuValAla                              70758085                                                                      CTCTACACCCGGGACGAGCACTTTGCCATCGCGGCGGACAGCGAGGCC883                           LeuTyrThrArgAspGluHisPheAlaIleAlaAlaAspSerGluAla                              9095100                                                                       GAGCAAGACAGCTGGTACCAGGCTCTCCTACAGCTGCACAACCGTGCT931                           GluGlnAspSerTrpTyrGlnAlaLeuLeuGlnLeuHisAsnArgAla                              105110115                                                                     AAGGGCCACCACGACGGAGCTGCGGCCCTCGGGGCGGGAGGTGGTGGT979                           LysGlyHisHisAspGlyAlaAlaAlaLeuGlyAlaGlyGlyGlyGly                              120125130                                                                     GGGGGCAGCTGCAGCGGCAGCTCCGGCCTTGGTGAGGCTGGGGAGGAC1027                          GlyGlySerCysSerGlySerSerGlyLeuGlyGluAlaGlyGluAsp                              135140145                                                                     TTGAGCTACGGTGACGTGCCCCCAGGACCCGCATTCAAAGAGGTCTGG1075                          LeuSerTyrGlyAspValProProGlyProAlaPheLysGluValTrp                              150155160165                                                                  CAAGTGATCCTGAAGCCCAAGGGCCTGGGTCAGACAAAGAACCTGATT1123                          GlnValIleLeuLysProLysGlyLeuGlyGlnThrLysAsnLeuIle                              170175180                                                                     GGTATCTACCGCCTTTGCCTGACCAGCAAGACCATCAGCTTCGTGAAG1171                          GlyIleTyrArgLeuCysLeuThrSerLysThrIleSerPheValLys                              185190195                                                                     CTGAACTCGGAGGCAGCGGCCGTGGTGCTGCAGCTGATGAACATCAGG1219                          LeuAsnSerGluAlaAlaAlaValValLeuGlnLeuMetAsnIleArg                              200205210                                                                     CGCTGTGGCCACTCGGAAAACTTCTTCTTCATCGAGGTGGGCCGTTCT1267                          ArgCysGlyHisSerGluAsnPhePhePheIleGluValGlyArgSer                              215220225                                                                     GCCGTGACGGGGCCCGGGGAGTTCTGGATGCAGGTGGATGACTCTGTG1315                          AlaValThrGlyProGlyGluPheTrpMetGlnValAspAspSerVal                              230235240245                                                                  GTGGCCCAGAACATGCACGAGACCATCCTGGAGGCCATGCGGGCCATG1363                          ValAlaGlnAsnMetHisGluThrIleLeuGluAlaMetArgAlaMet                              250255260                                                                     AGCGATGAGTTCCGCCCTCGCAGCAAGAGCCAGTCCTCGTCCAACTGC1411                          SerAspGluPheArgProArgSerLysSerGlnSerSerSerAsnCys                              265270275                                                                     TCTAACCCCATCAGCGTCCCCCTGCGCCGGCACCATCTCAACAATCCC1459                          SerAsnProIleSerValProLeuArgArgHisHisLeuAsnAsnPro                              280285290                                                                     CCGCCCAGCCAGGTGGGGCTGACCCGCCGATCACGCACTGAGAGCATC1507                          ProProSerGlnValGlyLeuThrArgArgSerArgThrGluSerIle                              295300305                                                                     ACCGCCACCTCCCCGGCCAGCATGGTGGGCGGGAAGCCAGGCTCCTTC1555                          ThrAlaThrSerProAlaSerMetValGlyGlyLysProGlySerPhe                              310315320325                                                                  CGTGTCCGCGCCTCCAGTGACGGCGAAGGCACCATGTCCCGCCCAGCC1603                          ArgValArgAlaSerSerAspGlyGluGlyThrMetSerArgProAla                              330335340                                                                     TCGGTGGACGGCAGCCCTGTGAGTCCCAGCACCAACAGAACCCACGCC1651                          SerValAspGlySerProValSerProSerThrAsnArgThrHisAla                              345350355                                                                     CACCGGCATCGGGGCAGGGCCCGGCTGCACCCCCCGCTCAACCACAGC1699                          HisArgHisArgGlyArgAlaArgLeuHisProProLeuAsnHisSer                              360365370                                                                     CGCTCCATCCCCATGCCGGCTTCCCGCTGCTCCCGTTCGGCCACCAGC1747                          ArgSerIleProMetProAlaSerArgCysSerArgSerAlaThrSer                              375380385                                                                     CCGGTCAGTCTGTCGTCCAGTAGCACCAGTGGCCATGGCTCCACCTCG1795                          ProValSerLeuSerSerSerSerThrSerGlyHisGlySerThrSer                              390395400405                                                                  GATTGTCTCTTCCCACGGCGATCTAGTGCTTCGGTGTCTGGTTCCCCC1843                          AspCysLeuPheProArgArgSerSerAlaSerValSerGlySerPro                              410415420                                                                     AGCGATGGCGGTTTCATCTCCTCGGATGAGTATGGCTCCAGTCCCTGC1891                          SerAspGlyGlyPheIleSerSerAspGluTyrGlySerSerProCys                              425430435                                                                     GATTTCCGGAGTTCCTTCCGCAGTGTCACTCCGGATTCCCTGGGCCAC1939                          AspPheArgSerSerPheArgSerValThrProAspSerLeuGlyHis                              440445450                                                                     ACCCCACCAGCCCGCGGTGAGGAGGAGCTAAGCAACTATATCTGCATG1987                          ThrProProAlaArgGlyGluGluGluLeuSerAsnTyrIleCysMet                              455460465                                                                     GGTGGCAAGGGGCCCTCCACCCTGACCGCCCCCAACGGTCACTACATT2035                          GlyGlyLysGlyProSerThrLeuThrAlaProAsnGlyHisTyrIle                              470475480485                                                                  TTGTCTCGGGGTGGCAATGGCCACCGCTGCACCCCAGGAACAGGCTTG2083                          LeuSerArgGlyGlyAsnGlyHisArgCysThrProGlyThrGlyLeu                              490495500                                                                     GGCACGAGTCCAGCCTTGGCTGGGGATGAAGCAGCCAGTGCTGCAGAT2131                          GlyThrSerProAlaLeuAlaGlyAspGluAlaAlaSerAlaAlaAsp                              505510515                                                                     CTGGATAATCGGTTCCGAAAGAGAACTCACTCGGCAGGCACATCCCCT2179                          LeuAspAsnArgPheArgLysArgThrHisSerAlaGlyThrSerPro                              520525530                                                                     ACCATTACCCACCAGAAGACCCCGTCCCAGTCCTCAGTGGCTTCCATT2227                          ThrIleThrHisGlnLysThrProSerGlnSerSerValAlaSerIle                              535540545                                                                     GAGGAGTACACAGAGATGATGCCTGCCTACCCACCAGGAGGTGGCAGT2275                          GluGluTyrThrGluMetMetProAlaTyrProProGlyGlyGlySer                              550555560565                                                                  GGAGGCCGACTGCCGGGACACAGGCACTCCGCCTTCGTGCCCACCCGC2323                          GlyGlyArgLeuProGlyHisArgHisSerAlaPheValProThrArg                              570575580                                                                     TCCTACCCAGAGGAGGGTCTGGAAATGCACCCCTTGGAGCGTCGGGGG2371                          SerTyrProGluGluGlyLeuGluMetHisProLeuGluArgArgGly                              585590595                                                                     GGGCACCACCGCCCAGACAGCTCCACCCTCCACACGGATGATGGCTAC2419                          GlyHisHisArgProAspSerSerThrLeuHisThrAspAspGlyTyr                              600605610                                                                     ATGCCCATGTCCCCAGGGGTGGCCCCAGTGCCCAGTGGCCGAAAGGGC2467                          MetProMetSerProGlyValAlaProValProSerGlyArgLysGly                              615620625                                                                     AGTGGAGACTATATGCCCATGAGCCCCAAGAGCGTATCTGCCCCACAG2515                          SerGlyAspTyrMetProMetSerProLysSerValSerAlaProGln                              630635640645                                                                  CAGATCATCAATCCCATCAGACGCCATCCCCAGAGAGTGGACCCCAAT2563                          GlnIleIleAsnProIleArgArgHisProGlnArgValAspProAsn                              650655660                                                                     GGCTACATGATGATGTCCCCCAGCGGTGGCTGCTCTCCTGACATTGGA2611                          GlyTyrMetMetMetSerProSerGlyGlyCysSerProAspIleGly                              665670675                                                                     GGTGGCCCCAGCAGCAGCAGCAGCAGCAGCAACGCCGTCCCTTCCGGG2659                          GlyGlyProSerSerSerSerSerSerSerAsnAlaValProSerGly                              680685690                                                                     ACCAGCTATGGAAAGCTGTGGACAAACGGGGTAGGGGGCCACCACTCT2707                          ThrSerTyrGlyLysLeuTrpThrAsnGlyValGlyGlyHisHisSer                              695700705                                                                     CATGTCTTGCCTCACCCCAAACCCCCAGTGGAGAGCAGCGGTGGTAAG2755                          HisValLeuProHisProLysProProValGluSerSerGlyGlyLys                              710715720725                                                                  CTCTTACCTTGCACAGGTGACTACATGAACATGTCACCAGTGGGGGAC2803                          LeuLeuProCysThrGlyAspTyrMetAsnMetSerProValGlyAsp                              730735740                                                                     TCCAACACCAGCAGCCCCTCCGACTGCTACTACGGCCCTGAGGACCCC2851                          SerAsnThrSerSerProSerAspCysTyrTyrGlyProGluAspPro                              745750755                                                                     CAGCACAAGCCAGTCCTCTCCTACTACTCATTGCCAAGATCCTTTAAG2899                          GlnHisLysProValLeuSerTyrTyrSerLeuProArgSerPheLys                              760765770                                                                     CACACCCAGCGCCCCGGGGAGCCGGAGGAGGGTGCCCGGCATCAGCAC2947                          HisThrGlnArgProGlyGluProGluGluGlyAlaArgHisGlnHis                              775780785                                                                     CTCCGCCTTTCCACTAGCTCTGGTGGCCTTCTCTATGCTGCAACAGCA2995                          LeuArgLeuSerThrSerSerGlyGlyLeuLeuTyrAlaAlaThrAla                              790795800805                                                                  GATGATTCTTCCTCTTCCACCAGCAGCGACAGCCTGGGTGGGGGATAC3043                          AspAspSerSerSerSerThrSerSerAspSerLeuGlyGlyGlyTyr                              810815820                                                                     TGCGGGGCTAGGCTGGAGCCCAGCCTTCCACATCCCCACCATCAGGTT3091                          CysGlyAlaArgLeuGluProSerLeuProHisProHisHisGlnVal                              825830835                                                                     CTGCAGCCCCATCTGCCTCGAAAGGTGGACACAGCTGCTCAGACCAAT3139                          LeuGlnProHisLeuProArgLysValAspThrAlaAlaGlnThrAsn                              840845850                                                                     AGCCGCCTGGCCCGGCCCACGAGGCTGTCCCTGGGGGATCCCAAGGCC3187                          SerArgLeuAlaArgProThrArgLeuSerLeuGlyAspProLysAla                              855860865                                                                     AGCACCTTACCTCGGGCCCGAGAGCAGCAGCAGCAGCAGCAGCCCTTG3235                          SerThrLeuProArgAlaArgGluGlnGlnGlnGlnGlnGlnProLeu                              870875880885                                                                  CTGCACCCTCCAGAGCCCAAGAGCCCGGGGGAATATGTCAATATTGAA3283                          LeuHisProProGluProLysSerProGlyGluTyrValAsnIleGlu                              890895900                                                                     TTTGGGAGTGATCAGTCTGGCTACTTGTCTGGCCCGGTGGCTTTCCAC3331                          PheGlySerAspGlnSerGlyTyrLeuSerGlyProValAlaPheHis                              905910915                                                                     AGCTCACCTTCTGTCAGGTGTCCATCCCAGCTCCAGCCAGCTCCCAGA3379                          SerSerProSerValArgCysProSerGlnLeuGlnProAlaProArg                              920925930                                                                     GAGGAAGAGACTGGCACTGAGGAGTACATGAAGATGGACCTGGGGCCG3427                          GluGluGluThrGlyThrGluGluTyrMetLysMetAspLeuGlyPro                              935940945                                                                     GGCCGGAGGGCAGCCTGGCAGGAGAGCACTGGGGTCGAGATGGGCAGA3475                          GlyArgArgAlaAlaTrpGlnGluSerThrGlyValGluMetGlyArg                              950955960965                                                                  CTGGGCCCTGCACCTCCCAGGGCTGCTAGCATTTGCAGGCCTACCCGG3523                          LeuGlyProAlaProProArgAlaAlaSerIleCysArgProThrArg                              970975980                                                                     GCAGTGCCCAGCAGCCGGGGTGACTACATGACCATGCAGATGAGTTGT3571                          AlaValProSerSerArgGlyAspTyrMetThrMetGlnMetSerCys                              985990995                                                                     CCCCGTCAGAGCTACGTGGACACCTCGCCAGCTGCCCCTGTAAGCTAT3619                          ProArgGlnSerTyrValAspThrSerProAlaAlaProValSerTyr                              100010051010                                                                  GCTGACATGCGAACAGGCATTGCTGCAGAGGAGGTGAGCCTGCCCAGG3667                          AlaAspMetArgThrGlyIleAlaAlaGluGluValSerLeuProArg                              101510201025                                                                  GCCACCATGGCTGCTGCCTCCTCATCCTCAGCAGCCTCTGCTTCCCCG3715                          AlaThrMetAlaAlaAlaSerSerSerSerAlaAlaSerAlaSerPro                              1030103510401045                                                              ACTGGGCCTCAAGGGGCAGCAGAGCTGGCTGCCCACTCGTCCCTGCTG3763                          ThrGlyProGlnGlyAlaAlaGluLeuAlaAlaHisSerSerLeuLeu                              105010551060                                                                  GGGGGCCCACAAGGACCTGGGGGCATGAGCGCCTTCACCCGGGTGAAC3811                          GlyGlyProGlnGlyProGlyGlyMetSerAlaPheThrArgValAsn                              106510701075                                                                  CTCAGTCCTAACCGCAACCAGAGTGCCAAAGTGATCCGTGCAGACCCA3859                          LeuSerProAsnArgAsnGlnSerAlaLysValIleArgAlaAspPro                              108010851090                                                                  CAAGGGTGCCGGCGGAGGCATAGCTCCGAGACTTTCTCCTCAACACCC3907                          GlnGlyCysArgArgArgHisSerSerGluThrPheSerSerThrPro                              109511001105                                                                  AGTGCCACCCGGGTGGGCAACACAGTGCCCTTTGGAGCGGGGGCAGCA3955                          SerAlaThrArgValGlyAsnThrValProPheGlyAlaGlyAlaAla                              1110111511201125                                                              GTAGGGGGCGGTGGCGGTAGCAGCAGCAGCAGCGAGGATGTGAAACGC4003                          ValGlyGlyGlyGlyGlySerSerSerSerSerGluAspValLysArg                              113011351140                                                                  CACAGCTCTGCTTCCTTTGAGAATGTGTGGCTGAGGCCTGGGGAGCTT4051                          HisSerSerAlaSerPheGluAsnValTrpLeuArgProGlyGluLeu                              114511501155                                                                  GGGGGAGCCCCCAAGGAGCCAGCCAAACTGTGTGGGGCTGCTGGGGGT4099                          GlyGlyAlaProLysGluProAlaLysLeuCysGlyAlaAlaGlyGly                              116011651170                                                                  TTGGAGAATGGTCTTAACTACATAGACCTGGATTTGGTCAAGGACTTC4147                          LeuGluAsnGlyLeuAsnTyrIleAspLeuAspLeuValLysAspPhe                              117511801185                                                                  AAACAGTGCCCTCAGGAGTGCACCCCTGAACCGCAGCCTCCCCCACCC4195                          LysGlnCysProGlnGluCysThrProGluProGlnProProProPro                              1190119512001205                                                              CCACCCCCTCATCAACCCCTGGGCAGCGGTGAGAGCAGCTCCACCCGC4243                          ProProProHisGlnProLeuGlySerGlyGluSerSerSerThrArg                              121012151220                                                                  CGCTCAAGTGAGGATTTAAGCGCCTATGCCAGCATCAGTTTCCAGAAG4291                          ArgSerSerGluAspLeuSerAlaTyrAlaSerIleSerPheGlnLys                              122512301235                                                                  CAGCCAGAGGACCGTCAGTAGCTCAACTGGACATCACAGCAGGTCGTT4339                          GlnProGluAspArgGln                                                            1240                                                                          TCATGGTGACAAAGTCAGAAGACAAAACTGCTTTTAACCTTGTCCTTGAATTCTGTTCTT4399              CGCCTCTGCCCCTTCCTGTTCTTTCCCACTGCTTCCTCAGGGAGAATGCACTTACATTCT4459              CAGGGCATACAAGATGCTCACCCACACTGACATTGGCAGAGAGTCAAACAAACATGTAGG4519              AGCAGCCACAGGAGGGCTTTTTCGTTTGAGGAATTCCCAAGTGAAGTAGTTACTGCAGTA4579              TTTTTAAACATATATCCTATGCCAGTTCTGCGTTTTGTAGAGTTCCTCCGTAAGAAGCTT4639              GATTTGTTTGTTGAAGTTTTCTTTTCACTATATATTTAGGTCAGCCCCTGGAAGGACAGT4699              TCTACAAAAAATACCCGTTAACACAGGGGCTAAACCCTTCCTTATCTTAAACTATCTTAA4759              TAGTTTCTGGGAGCCCTTAAGGGTGATCTTATCAAGTTGTTCTCTGTACTTTTGTTCTGT4819              GATTTCATAATACTAGGGCAACATAAACAGCAGCGGGAAGCATTGATTTCTATTCATCCT4879              GCCCTAAAAAGATCAGGAGTAAGAGCTTTTTAGAAATATGTATTTAGAGAGAAGTACCTA4939              TCTATTTTGTGATCTCTCAAGAAAGTAATTATGGGTGACGTTCTCCTTTTGTTCATGTAC4999              CAGGATTTGTGAAATATTATTCACACAACCGACCCACCATCCCACGGGCCTGGCCTCTCT5059              TGTACAGGATATGCAGGAAACTCTGTATGTGTCTGGGACCCATTATTAAGAGTTATGGGA5119              GTTCATCCTAGGATGTCTGCCTTATAGTTATCTCTTCTTCTTGCACTGAGACATTACAGA5179              TATCATTTGGGGGCTACTATATATCTTCTGTAAAATTACTTTTATTTGTTGAAGAAGAAT5239              GCATACTAAGTCAGGAACATGCCTTAATTTGTTTTGTTTTGCAATTGAGTAGAAGGGCTA5299              AACTGTATCCCTCCACTTTTAGGGTTATTTGCCTGTGTGCCTTTAAGTTCAAAAGTAGAC5359              ACCACAGTAAATGCTGAAAGTTGGCTTTAGGTCTTCTGTGGCTAATGCCGTATTAAAAAT5419              GAAAAACATTTGTGGTAGAAATTAGCCTGCCCTTCGTTCTGTTGATCCTGTTTTCTGGTG5479              GTCATAATTGTGGGTAGAAGAGAGTACAGTTTGCAAATAATGTGATGAGTTGGCAATGCA5539              GAAGTTTCCAGCATTTGGAAACACTTTACTCTGACAAACTGATTATCTTGTGAATTTTAT5599              CTATGCTCCACAGAATGAGCTTTTAAAAGCACTGATTTTTCTTAATTTGTGTCCATTCAT5659              AAGAAATTAATCTGTGCCCTGGTTTCCTATTGACAGGTATTTATTTATCATGTGTTCATA5719              GTCTTCTTAATTCTGTTTCCAATATTTGATCCATATAATTCTCTATTTTATAAAGCAAGA5779              AAAAGGTATATGAACACTCAAATGAAGATTTTGGGTGATATGTTACAAAAAGCATTTATT5839              TGATCAGTATTTACTTCAACATTTATTTTCATCATTCACTAGAAGAAAGATTTAATTGTG5899              TATATCAACATCAGTAGTACAAATCTTGTTATATCAAATGATGTTTTTGGGAGTTCAGAA5959              TCCCTCAACACTTTAAGCATTTGTATTATAAAGTGCCTCATTGGTAAAATAATGAGAATT6019              TGAAGAAAACCAGCCCAGCAGAACTAAAATTTTGGTTTTAAAGGAGATAAAGAGAATAAG6079              TTTTTCTTACTTGTCATCTTAATTTGTTTAGGTTTCTTTTTATAGAGTAGAATAAATGAT6139              GTTTGCTCTGAAG6152                                                             (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1243 amino acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      MetAlaSerProProGluSerAspGlyPheSerAspValArgLysVal                              151015                                                                        GlyTyrLeuArgLysProLysSerMetHisLysArgPhePheValLeu                              202530                                                                        ArgAlaAlaSerGluAlaGlyGlyProAlaArgLeuGluTyrTyrGlu                              354045                                                                        AsnGluLysLysTrpArgHisLysSerSerAlaProLysArgSerIle                              505560                                                                        ProLeuGluSerCysPheAsnIleAsnLysArgAlaAspSerLysAsn                              65707580                                                                      LysHisLeuValAlaLeuTyrThrArgAspGluHisPheAlaIleAla                              859095                                                                        AlaAspSerGluAlaGluGlnAspSerTrpTyrGlnAlaLeuLeuGln                              100105110                                                                     LeuHisAsnArgAlaLysGlyHisHisAspGlyAlaAlaAlaLeuGly                              115120125                                                                     AlaGlyGlyGlyGlyGlyGlySerCysSerGlySerSerGlyLeuGly                              130135140                                                                     GluAlaGlyGluAspLeuSerTyrGlyAspValProProGlyProAla                              145150155160                                                                  PheLysGluValTrpGlnValIleLeuLysProLysGlyLeuGlyGln                              165170175                                                                     ThrLysAsnLeuIleGlyIleTyrArgLeuCysLeuThrSerLysThr                              180185190                                                                     IleSerPheValLysLeuAsnSerGluAlaAlaAlaValValLeuGln                              195200205                                                                     LeuMetAsnIleArgArgCysGlyHisSerGluAsnPhePhePheIle                              210215220                                                                     GluValGlyArgSerAlaValThrGlyProGlyGluPheTrpMetGln                              225230235240                                                                  ValAspAspSerValValAlaGlnAsnMetHisGluThrIleLeuGlu                              245250255                                                                     AlaMetArgAlaMetSerAspGluPheArgProArgSerLysSerGln                              260265270                                                                     SerSerSerAsnCysSerAsnProIleSerValProLeuArgArgHis                              275280285                                                                     HisLeuAsnAsnProProProSerGlnValGlyLeuThrArgArgSer                              290295300                                                                     ArgThrGluSerIleThrAlaThrSerProAlaSerMetValGlyGly                              305310315320                                                                  LysProGlySerPheArgValArgAlaSerSerAspGlyGluGlyThr                              325330335                                                                     MetSerArgProAlaSerValAspGlySerProValSerProSerThr                              340345350                                                                     AsnArgThrHisAlaHisArgHisArgGlyArgAlaArgLeuHisPro                              355360365                                                                     ProLeuAsnHisSerArgSerIleProMetProAlaSerArgCysSer                              370375380                                                                     ArgSerAlaThrSerProValSerLeuSerSerSerSerThrSerGly                              385390395400                                                                  HisGlySerThrSerAspCysLeuPheProArgArgSerSerAlaSer                              405410415                                                                     ValSerGlySerProSerAspGlyGlyPheIleSerSerAspGluTyr                              420425430                                                                     GlySerSerProCysAspPheArgSerSerPheArgSerValThrPro                              435440445                                                                     AspSerLeuGlyHisThrProProAlaArgGlyGluGluGluLeuSer                              450455460                                                                     AsnTyrIleCysMetGlyGlyLysGlyProSerThrLeuThrAlaPro                              465470475480                                                                  AsnGlyHisTyrIleLeuSerArgGlyGlyAsnGlyHisArgCysThr                              485490495                                                                     ProGlyThrGlyLeuGlyThrSerProAlaLeuAlaGlyAspGluAla                              500505510                                                                     AlaSerAlaAlaAspLeuAspAsnArgPheArgLysArgThrHisSer                              515520525                                                                     AlaGlyThrSerProThrIleThrHisGlnLysThrProSerGlnSer                              530535540                                                                     SerValAlaSerIleGluGluTyrThrGluMetMetProAlaTyrPro                              545550555560                                                                  ProGlyGlyGlySerGlyGlyArgLeuProGlyHisArgHisSerAla                              565570575                                                                     PheValProThrArgSerTyrProGluGluGlyLeuGluMetHisPro                              580585590                                                                     LeuGluArgArgGlyGlyHisHisArgProAspSerSerThrLeuHis                              595600605                                                                     ThrAspAspGlyTyrMetProMetSerProGlyValAlaProValPro                              610615620                                                                     SerGlyArgLysGlySerGlyAspTyrMetProMetSerProLysSer                              625630635640                                                                  ValSerAlaProGlnGlnIleIleAsnProIleArgArgHisProGln                              645650655                                                                     ArgValAspProAsnGlyTyrMetMetMetSerProSerGlyGlyCys                              660665670                                                                     SerProAspIleGlyGlyGlyProSerSerSerSerSerSerSerAsn                              675680685                                                                     AlaValProSerGlyThrSerTyrGlyLysLeuTrpThrAsnGlyVal                              690695700                                                                     GlyGlyHisHisSerHisValLeuProHisProLysProProValGlu                              705710715720                                                                  SerSerGlyGlyLysLeuLeuProCysThrGlyAspTyrMetAsnMet                              725730735                                                                     SerProValGlyAspSerAsnThrSerSerProSerAspCysTyrTyr                              740745750                                                                     GlyProGluAspProGlnHisLysProValLeuSerTyrTyrSerLeu                              755760765                                                                     ProArgSerPheLysHisThrGlnArgProGlyGluProGluGluGly                              770775780                                                                     AlaArgHisGlnHisLeuArgLeuSerThrSerSerGlyGlyLeuLeu                              785790795800                                                                  TyrAlaAlaThrAlaAspAspSerSerSerSerThrSerSerAspSer                              805810815                                                                     LeuGlyGlyGlyTyrCysGlyAlaArgLeuGluProSerLeuProHis                              820825830                                                                     ProHisHisGlnValLeuGlnProHisLeuProArgLysValAspThr                              835840845                                                                     AlaAlaGlnThrAsnSerArgLeuAlaArgProThrArgLeuSerLeu                              850855860                                                                     GlyAspProLysAlaSerThrLeuProArgAlaArgGluGlnGlnGln                              865870875880                                                                  GlnGlnGlnProLeuLeuHisProProGluProLysSerProGlyGlu                              885890895                                                                     TyrValAsnIleGluPheGlySerAspGlnSerGlyTyrLeuSerGly                              900905910                                                                     ProValAlaPheHisSerSerProSerValArgCysProSerGlnLeu                              915920925                                                                     GlnProAlaProArgGluGluGluThrGlyThrGluGluTyrMetLys                              930935940                                                                     MetAspLeuGlyProGlyArgArgAlaAlaTrpGlnGluSerThrGly                              945950955960                                                                  ValGluMetGlyArgLeuGlyProAlaProProArgAlaAlaSerIle                              965970975                                                                     CysArgProThrArgAlaValProSerSerArgGlyAspTyrMetThr                              980985990                                                                     MetGlnMetSerCysProArgGlnSerTyrValAspThrSerProAla                              99510001005                                                                   AlaProValSerTyrAlaAspMetArgThrGlyIleAlaAlaGluGlu                              101010151020                                                                  ValSerLeuProArgAlaThrMetAlaAlaAlaSerSerSerSerAla                              1025103010351040                                                              AlaSerAlaSerProThrGlyProGlnGlyAlaAlaGluLeuAlaAla                              104510501055                                                                  HisSerSerLeuLeuGlyGlyProGlnGlyProGlyGlyMetSerAla                              106010651070                                                                  PheThrArgValAsnLeuSerProAsnArgAsnGlnSerAlaLysVal                              107510801085                                                                  IleArgAlaAspProGlnGlyCysArgArgArgHisSerSerGluThr                              109010951100                                                                  PheSerSerThrProSerAlaThrArgValGlyAsnThrValProPhe                              1105111011151120                                                              GlyAlaGlyAlaAlaValGlyGlyGlyGlyGlySerSerSerSerSer                              112511301135                                                                  GluAspValLysArgHisSerSerAlaSerPheGluAsnValTrpLeu                              114011451150                                                                  ArgProGlyGluLeuGlyGlyAlaProLysGluProAlaLysLeuCys                              115511601165                                                                  GlyAlaAlaGlyGlyLeuGluAsnGlyLeuAsnTyrIleAspLeuAsp                              117011751180                                                                  LeuValLysAspPheLysGlnCysProGlnGluCysThrProGluPro                              1185119011951200                                                              GlnProProProProProProProHisGlnProLeuGlySerGlyGlu                              120512101215                                                                  SerSerSerThrArgArgSerSerGluAspLeuSerAlaTyrAlaSer                              122012251230                                                                  IleSerPheGlnLysGlnProGluAspArgGln                                             12351240                                                                      (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 base pairs                                                      (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CCNGG5                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GCTCAGCGTTGGTGGTGGCGGTGG24                                                    (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GCCCGCTTGTTGATGTTGAAGCAGC25                                                   (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GAAGTGGCGGCACAAGTCGAGCGC24                                                    (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CCAGCCTCACCAAGGCCGGAGC22                                                      (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       ACCGTGCTAAGGGCCACCACGACG24                                                    (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       AGCACCACGGCCGCTGCCTCC21                                                       (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      TCTACCGCCTTTGCCTGACCAGC23                                                     (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GCAGTTGGACGAGGACTGGC20                                                        (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      ACCATCCTGGAGGCCATGCGG21                                                       (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      AGGGCTGCCGTCCACCGAGGCTGG24                                                    (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      CAGGCTCCTTCCGTGTCCGCG21                                                       (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GAAGCACTAGATCGCCGTGGG21                                                       (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      CTGTCGTCCAGTAGCACCAGTGG23                                                     (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GACCGTTGGGGGCGGTCAGGG21                                                       (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      GCGGTGAGGAGGAGCTAAGC20                                                        (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      GCCACTGAGGACTGGGACGGG21                                                       (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      AGAGAACTCACTCGGCAGGC20                                                        (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      GGCATGTAGCCATCATCCGTGTGG24                                                    (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      ACCCCTTGGAGCGTCGGGGG20                                                        (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      GCTGGGGCCACCTCCTAAGTCAGG24                                                    (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      CAGAGAGTGGACCCCAATGG20                                                        (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      GGCTGCTGGTGTTGGAGTCC20                                                        (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      TCTTGCCTCACCCCAAACCC20                                                        (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      ATAGAGAAGGCGACCAGAGC20                                                        (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      GAGCCGGAGGAGGGTGCCCG20                                                        (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      AGGTAAGGTGCTGGCCTTGG20                                                        (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      CAGACCAATAGCCGCCTGGC20                                                        (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      TTCATGTACTCCTCAGTGCC20                                                        (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      CTTCTGTCAGGTGTCCATCC20                                                        (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      TGGCGAGGTGTCCACGTAGC20                                                        (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      GGGCAGTGCCCAGCAGCCGG20                                                        (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      CCAGCAGGGACGAGTGGGCAGC22                                                      (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      CCTCAGCAGCCTCTGCTTCC20                                                        (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      CCACCGCCCCCTACTGCTGC20                                                        (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      CCACACCCAGTGCCACCCGG20                                                        (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      GAGGGCACTGTTTGAAGTCC20                                                        (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      GAGCCAGCCAAACTGTGTGG20                                                        (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      AACGACCTGCTGTGATGTCC20                                                        __________________________________________________________________________

We claim:
 1. An isolated DNA sequence comprising the sequence of SEQ IDNO:1 encoding insulin receptor substrate 1 (IRS-1), the DNA sequencecontaining a mutation of at least one nucleotide selected from the groupconsisting of a mutation of G to A in the first position of codon 972, amutation of A to G in the third position of codon 805, and a mutation ofG to C in the third position of codon 894, wherein the mutationinterferes with signal transduction through IRS-1.
 2. The DNA sequenceof claim 1, wherein the mutation gives rise to at least one amino acidsubstitution in the IRS-1 sequence.
 3. The DNA sequence of claim 1comprising a mutation of G to A at nucleotide 3494 of SEQ ID NO:1.
 4. Arecombinant expression vector comprising the DNA sequence of claim
 1. 5.An isolated mammalian cell containing and expressing the DNA sequence ofclaim
 3. 6. An isolated IRS-1 protein containing at least one amino acidsubstitution wherein glycine is substituted by arginine at position 972in SEQ ID NO:2 and wherein said substitution interferes with signaltransduction through IRS-1.